In the invention described, the terms "antioxidant (or reducing or reductive) capacity, antioxidant (or reducing or reductive) power, antioxidant (or reducing or reductive) ability, antioxidant (or reductive or reducing)" are held to be synonymous and interchangeable. Oxidising species, or reactive oxidant species, are known to contribute to the cause of a number human diseases. In situations or conditions where antioxidant defence is inadequate, there may be oxidative changes to DNA, protein and lipids, which may lead to various degenerative diseases, such as coronary hear disease, cancer, diabetes, arthritis and cataracts (Gey K. F., Br. Med. Bull., (1993), 49, 679-99; Steinberg D., Circulation, (1992), 85, 2338-44; Cutteridge J. M. C., (1994) 91, 133-40; Gey K. F. et al, Am. J. Clin. Nutr., (1993), 57, 787S-797S).
Measurement of antioxidant status of biological fluids and tissues may be useful in the study of pro-oxidant:antioxidant balance and the risk of, or defence against, diseases associated with oxidative damage. The relative contributions to this by individual antioxidants, and assessment of the potential for ingestion or treatment with foodstuffs, pharmaceuticals, vitamin preparations, herbs etc., to improve antioxidant status may be useful in helping to plan risk reduction/health promoting strategies.
The term "antioxidant," can be employed as a generic term describing a group of compounds which may prevent the generation of oxidising species, remove such species or inactivate them, thus preventing, delaying or minimising oxidative changes to important biomolecules. Members of this group are diverse in structure and action, and include metal chelating compounds and enzymes such as catalase and superoxide dismutase. However, one important antioxidant sub-group comprises electron donating (i.e. reducing) antioxidant, such as (but not limited to) ascorbic acid (vitamin C), alpha, beta, gamma and delta tocopherols and tocotrienols (collectively known as "vitamin E"), uric acid, bilirubin, phenolic group compounds such as flavonoids, and thiol group-containing compounds such as protein and glutathione. Electron donating antioxidants, also known as scavenging and chain breaking antioxidants, destroy or inactivate oxidising species by means of electron transfer from "antioxidant" to the oxidant. In this application, the term "antioxidant" is used hereafter to describe such electron donating antioxidants.
Up until now, most tests used for measuring the antioxidant power of a sample have measured the ability of the sample to withstand the oxidative effects of reactive species purposely generated in the reaction mixture. For example, depletion of antioxidants denoted by a change in signal, such as the rate of oxygen utilisation (Wayner, D. D. M. et al, Biochim. Biophys. Acta, (1987), 924, 408-419, or chemiluminescence (GB 2245062; Popov, I. N., Free Radicals Biol. Med., (1994), 17, 267-71; Whitehead, T. P., et al, anal. Chim, Acta., (1992), 266, 265-277; Lissi, E., Free Radicals Biol. Med., (1995), 16, 581-90). However, these methods require specialised equipment and can be time consuming. Therefore, these methods are difficult to sue and limited in their applications. As a result of the lack of an inexpensive, simple, rapid and efficient method of measuring individual antioxidant and/or total antioxidant (reducing) power of biological samples, there is insufficient data on the clinical utility of assessing antioxidant status in the various disorders thought to be associated with oxidative changes/oxidative stress.
Specific antioxidants, such as ascorbic acid, are known to play a major role in antioxidant defence. However, most methods of measuring the concentration of ascorbic acid in samples are non-specific, redox linked colourimetric methods (Pachla L.A. et al, J. Assoc. Anal. Chem., (1985), 68 1-12). A more specific method has been disclosed by Liu L. S., et al, J. Chrom., (1993), 612, 63-70, which requires use of HPLC. However, this method is both time consuming and expensive. Lewin G., et al, J. Biochem. Biophys. Meth., (1994), 28, 277-82, has recently disclosed a sensitive method of measuring ascorbic acid using a photochemiluminescence method, however this method requires specialised equipment. Moreover, owing to the time consuming nature of most methods used to date, pre-treatment of samples is usually needed in order to stabilise the ascorbic acid content.
Furthermore, it is time consuming, expensive and difficult to the point of impracticality to attempt to measure all possible constituent antioxidants within a heterogeneous sample, such as blood plasma, urine, plant material, foodstuffs etc. Therefore, a method suitable for the speedy, specific and sensitive measurement of individual antioxidant(s) and/or net, overall or total antioxidant power of a wide range of sample types would have considerable advantages over currently available methods. This would especially be true if such a method could be performed on samples with no or minimal pre-treatment. Such a method would facilitate observational, clinical and nutritional studies into antioxidant defence and the role of oxidative stress in the aetiology and severity of chronic disease.